DNA rearrangement, somatic mutation, and expression of immunoglobulin heavy chain (IgH) genes occur exclusively in B cells. During the last several years, my laboratory has identified and characterized cis and trans elements that are candidate regulators of VDJ joining, class switching and IgH gene expression. These include identification of a key B cell transcription factor, BSAP, and characterization of the IgH 3' regulatory region, which contains four enhancers. Only the most distal enhancer, hs4, appears to be active throughout B cell differentiation, as assessed in cultured cell lines. We have actively studied the extended 3' IgH region for its role in IgH gene replication, together with Roy Riblet and Carl Schildkraut. We find dramatic differences in usage of origins of DNA replication for the IgH gene cluster in cultured non-B cell lines and in B cell lines representing different stages of maturation. Changes in enhancer activation and origin usage are occurring within a single approximately 100 kb DNA segment. The hypothesis of this proposal is that in vivo, there is a temporal and developmental relationship between regulation of IgH 3' enhancers and usage of IgH origins of replication that involves a mechanistic interface. Our first goal is to determine the temporal and cell-type specific activity of hs4 as a transcriptional enhancer in vivo. We will generate mice with an hs4-regulated transgene linked to a GFP expression reporter. Mutational analysis of hs4 transcriptional activity will be carried out in cell lines. Our second goal is to locate and identify IgH origins of replication in non-B, B and plasma cell lines and to determine whether normal cells show similar utilization of specific IgH replication origins. A third goal is to determine whether a boundary for the IgH locus/ chromosomal domain is located at hs4, at the dominant downstream ori used in MEL, or in between. We will assess the chromatin configuration of the 100 kb region in which changes in enhancer activation and origin usage are both occurring.